TECHNIQUES

 

Below you will find a brief description of the main techniques we use in our lab. We use in vitro and in vivo models of degeneration and regeneration of the peripheral and central nervous system, as well as diverse techniques for analysis, from ultrastructural to behavioral ones.

Ex vivo degeneration assays in central and peripheral nervous system

To study axonal degeneration in central and peripheral nervous system ex vivo, optic and sciatic nerve segments are dissected out from laboratory mice of different strain and cultured in multi-well plates for different periods of time. Pharmacological treatments are easy to perform in this ex vivo platform, which retains important tissular components, including glial cells and extracellular matrix. After incubation, a variety of techniques are performed including biochemical, light microscopy and electron microscopy techniques.

Degeneration and regeneration of sensory neurons in vitro

To induce axonal degeneration (AD), dorsal root ganglion sensory neurons (DRGs) cultured in vitro, are mechanically or functionally damaged (mechanical transection or axonal transport inhibition). Readouts of AD used by immunofluorescence are microtubule and neurofilament integrity among others. To evaluate axonal regeneration in vitro, DRGs are cultured and after neurite extension, DRGs are axotomized and bright field images are taken daily. Regeneration is quantified measuring the distance from the cut edge to the tip of the longest neurites. Additional analyses, as growth cone morphology, are also performed.

Regeneration in the PNS, morphological and functional tests

Peripheral nerve regeneration studies are also performed in vivo, to study the process in a physiological context. After experimental nerve damage, axons are allowed to regenerate and morphologcal and functional studies are performed to evaluate nerve regeneration. By optic and electron microscopy, the extent of regeneration can be measured. Functionally, rotarod studies, sciatic nerve functional index (SFI) and the pinch test reflex response are performed to evaluate functional recovery after nerve damage.

Purification of extracellular vesicles from cultured cells

We purified vesicular vectors from cell cultures supernatants or plasma using different techniques: (i) serial centrifugation to eliminate cell debris and larger vesicles and then ultracentrifugation to pellet small vesicles called exosomes, (ii) immunoisolation of exosomes using latex beads conjugated to specific antibodies followed by FACS analysis, and (iii) sucrose gradients to purify and characterize exosomes according to their density. Purified exosomes are used for biochemical characterization, proteomics and genomic studies and to evaluate their effect in other recipient cells.

Spinal cord injury (SCI) in mice and rat

We are currently using two models of SCI. A lateral dorso-ventral hemisection at T13 in mice and a clip-compression model at T8 in rats, as a preclinical approach to evaluate the neuroprotective role of different pharmacological treatments. To assess the locomotor recovery, we use the BBB and BMS scores for rats and mice, respectively. We also perform morphological analysis at the light and electron microscope levels. We use antibody markers for neurons, oligodendrocytes, microglia and astrocyte, to evaluate their response after SCI. And perform studies in genetically modified mice for functional enquiries.

Tracing of neuronal projections by dyes and viral infection

The corticospinal tract in mice is traced by two methods. The classical Biotin-Dextran-Amine (BDA) injected in the sensorimotor cortex, and with a GFP-recombinant adeno-associated viral (AAV) vector. To visualize the BDA, we use either a fluorescent Avidin-Alexa 488 or classical DAB histochemical reaction. Injection of the tracer and viral particles is performed using a stereotaxic apparatus in order to inject sub-population of neurons located in specific brain regions.

Electron microscopy techniques

We have implemented several techniques for ultrastructural analysis in different tissues and culture platforms, including central and peripheral nervous system, cell lines and primary cultures in standard and multi-well dishes, campenot chambers and microfluid chambers. We perform standard scanning and transmission electron microscopy (Barrientos et al., J Neuroscience, 2011), as well as single (Vidal et al., Hum Mol Genet 2012) and double immunogold techniques (Hetz et al., Gene & Dev, 2009). Serial section is also performed in the lab. We have also implemented techniques for correlative light and electron microscopy (Castillo et al., EMBO J, 2012).

Teasing of individual nerve fibers from peripheral nerves

The peripheral nerve offers several advantages for experimental studies. Is readily accesible, then is relatively easy to manipulate in vivo by surgical techniques compared to the central nervous system. In addition analisis of individual nerve fibers, composed by the axon and its associated glial cell can be performed by immunofluorescence techniques after teasing individual fibers under a dissecting microsope. In this way a very detail morphological analysis can be performed using quantitative techniques (see Court et al., Nature 2004, Court et al., J Neuroscience 2008 and 2009).